We propose to build, characterize and validate an organotypic cell-based system that simulates the trabecular meshwork (TM) in organization and physiology. This is to provide a valid model for studying the tissue's cell and molecular biology within an environment comparable to that of the living eye. The cell/molecular basis of TM outflow regulation is difficult to decipher partly because the tissue's in situ organization is so complex and its cells inaccessible. The system will have the form of a flow apparatus divided into 2 chambers by a tissue cassette which houses a synthetic, flexible matrix. On the matrix, human TM cells will be grown to confluence. Fluid pressure in each chamber and across cells can be controlled to mimic intraocular pressure, episcleral venous pressure and the tissue's transcellular pressure gradient respectively, allowing fluid flow across the cell-matrix complex to be set at levels comensurate with normal physiology or otherwise. Initially, flow across cell monolayers grown on 2-D matrices will be measured. A more advanced model will include TM cells grown in 3-D matrices and co-cultured TM-Schlemm's canal cells to more closely mimic the TM's juxtacanalicular region. Flow will be determined at different hydraulic pressures, and compared and validated by comparable observations in live monkey eyes. Cell morphology and responses, especially involving the actin cytoskeleton and cell adhesions, will be characterized. Probing the system with agents such as H7 which are known to alter TM outflow will provide further validation and a firm basis for subsequently dissecting out the cell and molecular biology of the TM. Not only will the system allow the TM's biology to be studied in the context of the forces the tissue usually encounters, but cell behaviour can also be related back to tissue function. We expect that this will pave the way to a better understanding of the tissue's biology in health and disease. It will also provide a novel and cost-effective means of developing and testing new glaucoma therapies. [unreadable] [unreadable] [unreadable]